Taxane compounds, compositions and methods

ABSTRACT

The present invention provides a method for the preparation of orally available pentacyclic taxane compounds, as well as intermediates useful in their preparation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of U.S. patent applicationSer. No. 13/170,830, filed on Jun. 26, 2011, which claims the benefit ofU.S. Patent Application No. 61/360,135, filed on Jun. 30, 2010, thecontents of which are specifically incorporated by reference herein intheir entirety.

FIELD OF THE INVENTION

The present invention relates generally to methods for preparation oftaxane derivatives that have antitumor activity and can be orallyadministered, in particular pentacyclic taxanes.

BACKGROUND OF THE INVENTION

Taxol is a natural substance represented by the following chemicalstructural formula, which can be obtained in small amounts from the barkor other parts of Taxus brevifolia.

It is known that taxol has antitumor activity, and its mechanism ofaction is believed to be based on its ability to inhibitdepolymerization of microtubules during cell division. At the time ofthe discovery of taxol this mechanism of action was different from theconventional antitumor agents, so it became of great interest for itspotential clinical application as an antitumor agent.

Taxol can be obtained from natural sources, but only in very smallamounts. However, taxol derivatives can now be synthesized using a taxolprecursor, 10-O-deacetylbaccatine III (“10-DAB III”), which can beobtained from leaves and other parts of Taxus plants in relativelylarger amounts. One such taxol derivative, docetaxel, is marketed bySanofi under the tradename Taxotere® and has been approved for thetreatment of various cancers, including breast cancer.

Recently, in U.S. Pat. No. 6,646,123, inventors at DaiichiPharmaceutical Co. reported on a series of pentacyclic taxane compounds.These pentacyclic taxanes were obtained by reduction of the 9-positionketone of known taxanes to form a 9-position hydroxyl group which wasthen linked to the 10-position hydroxyl group to form a cyclic acetal.The resulting compounds have strong antitumor activity.

Additional studies on pentacyclic taxanes are reported in U.S. Pat. No.6,677,456 (Daiichi Sankyo). These compounds have oral antitumor activityand therefore the potential to eliminate the toxic side effectsassociated with the use of Cremophor EL (a polyoxyethylated castor oil)and polysorbate 80 to solubilize taxanes for intravenous administration.One such compound is tesetaxel, having the following structure.

There is a continuing need for efficient and cost-effective synthesisschemes for preparing orally available taxol derivatives, such astesetaxel, and for intermediates useful in such syntheses.

SUMMARY OF THE INVENTION

Accordingly, one aspect of the present; invention is directed to acompound represented by formula (Ia) and methods for preparing a taxane,including tesetaxel, comprising reacting the compound represented byformula (Ia)

with a taxane side chain precursor compound to couple the side chainprecursor compound to C13 of the compound represented by formula (Ia)

In a specific example, C13 of the compound represented by formula (Ia)is coupled with a side chain precursor compound represented by formula(II)

wherein R² is an alkoxy group having from 1 to 6 carbon atoms or ahalogen atom such as fluorine (F), bromine (Br), iodine (I) or chlorine(Cl) and is a protected hydroxyl group.

For synthesis of pentacyclic taxanes other than tesetaxel, thedimethylaminomethyl group of the compound represented by formula (Ia) isreplaced with any of the R4 and R5 substituents disclosed in U.S. Pat.No. 6,646,123, discussed above. In a specific embodiment thedimethylaminomethyl group is replaced with another amino-containinggroup such as morpholinomethyl.

In some embodiments, R³³ is triisopropylsilyl, while in others R³³ ismethoxy methylethoxy (also referred to as 2-methoxy propyloxy or MOP).

According to another aspect of the invention, the compound representedby formula (Ib) is provided. In one embodiment, the compound representedby formula (Ia) can be derived from the precursor compound representedby formula (Ib)

by reducing the C6-C7 double bond to a single bond.

Alternatively, the compound represented by formula (Ia) can be derivedfrom a precursor compound represented by formula (III)

by converting the terminal olefin (vinyl) group to an aldehyde andreacting the product aldehyde with an amine to form adimethylaminomethyl group.

Yet another aspect of the present invention provides a compoundrepresented by formula (VII) which is useful as an intermediate compoundin the synthesis of tesetaxel and other pentacyclic taxanes:

The compounds represented by formulas (Ia), (Ib), (III) and (IV) are allderivable from the compound represented by formula (VII) according tothe methods described herein.

Yet another aspect of the present invention provides a method for thepreparation of DOH which involves reduction of the C6-C7 double bond ofthe compound represented by formula (VII) to obtain DOH.

Yet another aspect of the present invention provides a compoundrepresented by formula (IX) which is useful as an intermediate compoundin the synthesis of tesetaxel end other pentacyclic taxanes:

The intermediate compounds represented by formulas (VII) and (XI) arederivable from the compound represented by formula (IX) as describedherein.

Yet another aspect of the invention provides a compound represented byformula (X) which is useful as an intermediate compound in the synthesisof tesetaxel and other pentacyclic taxanes:

The intermediate compounds represented by formulas (VII), (Ib) and (Ia)are all derivable from the compound represented by formula (X) asdescribed herein.

Another aspect of the present invention is directed to a compoundsrepresented by formula (XI) which is useful as an intermediate compoundin the synthesis of tesetaxel and other pentacyclic taxanes:

The compound represented by formula (XI) can be derived from thecompound represented by formula (IX) as described herein.

Another aspect of the present invention is directed to pharmaceuticallyacceptable acid addition salts of tesetaxel, including, for example,monobasic, dibasic, tribasic or polybasic acid salts. The salt forms oftesetaxel may comprise only tesetaxel and the acid, or they may behydrates and/or solvates of the acid addition salt of tesetaxel. Theseacid addition salts of tesetaxel are represented by the followingstructural formula:

(TT)_(m).(HX)_(n).(H₂O)_(y).(Sol)_(z)   (formula (XII))

-   -   wherein    -   TT is tesetaxel;    -   HX is a monobasic, dibasic, tribasic or polybasic acid;    -   Sol is an organic solvent;    -   m and n are each independently an integer from about 1 to about        5; and    -   y and z are each independently an integer from 0 to about 5.

The acid addition salts of tesetaxel may be comprised only of tesetaxeland the acid (i.e., y and z are both 0) or they may be hydrates and/orsolvates of the acid addition salt (i.e., y and z are each independentlyintegers from about 1 to about 5, y is 0 and z is an integer from about1 to about 5, or z is 0 and y is an integer from about 1 to about 5.).

In an additional aspect, the present invention provides methods oftreating cancer using the foregoing acid addition salts of tesetaxel. Insuch methods the acid addition salt is administered to a cancer patientin an amount effective to treat the cancer. Administration may be by anyappropriate route, including injection, infusion or oral administration.

The compounds and methods employed in the syntheses of the inventionprovide several advantages and improvements over prior art compounds andmethods for synthesis of Pentacyclic taxanes. First, active taxanesrequire handling and processing in high containment facilities due totheir potency and toxicity. Such specialized handling substantiallyincreases the cost of manufacture. The synthesis methods of theinvention decrease the amount of time and handling under highcontainment conditions by making attachment of the side chain the lastkey synthetic step of the method. As taxanes become active only when theside chain is attached, in the inventive process special handling isonly required for attachment of the side chain and purification of thefinal product. This substantially reduces the cost of taxanemanufacture.

In addition, by making coupling of the well-characterized, purified,specification-set side chain to the well-characterized, purified,specification-set pentacyclic core intermediate the last key step of thesynthesis a well characterized, highly purified specification-setproduct can be obtained reproducibly with better yield.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a reaction scheme for synthesis of apentacyclic taxane core structure, including alternative steps forsynthesis of intermediates.

FIG. 2 is an illustration of a general reaction scheme for synthesis ofa β-lactam intermediate for preparation of taxanes.

FIG. 3 is an illustration of a specific reaction scheme for synthesis ofthe compound represented by formula (Ia) and conversion of formula (Ia)to tesetaxel.

FIG. 4 is an illustration of an alternative specific reaction scheme forsynthesis of the compound represented by formula (Ia).

FIG. 5 is an illustration of an alternative reaction scheme for couplingthe compound represented by formula (Ia) to the side chain precursor toproduce tesetaxel.

FIG. 6 is an HPLC analysis of tesetaxel produced according to themethods of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Although the invention herein is described with reference to particularembodiments, it is to be understood that these embodiments are merelyillustrative of the principles and applications of the presentinvention. It is therefore to be understood that numerous modificationsmay be made to the illustrative embodiments and that other arrangementsmay be devised without departing from the spirit and scope of thepresent invention as defined by the appended claims.

As used herein, the designation “Me” means methyl, the designation “Bz”means benzoyl, the designation “Ac” means acetyl and the designation“Boc” means t-butoxycarbonyl.

As used herein, the term “derived” or “derivable” in connection withsynthesis of a compound from a precursor compound means that thecompound can be obtained by chemical synthesis from the identifiedprecursor, either directly in a single step or in a multi-step processstarting with the identified precursor compound.

One aspect of the present invention is directed to a method for thepreparation of tesetaxel. In U.S. Pat. No. 6,677,456, tesetaxel isprepared by coupling the side chain to C13 of a polycyclic taxane corecompound before completing synthesis of the tesetaxel fifth ring.

Applicants have found that a robust synthesis of pentacyclic taxanecompounds having a dimethylaminomethyl or other amino-containing groupin the fifth ring can be achieved by converting the vinyl group of thefifth ring precursor to the dimethylaminomethyl or otheramino-containing group prior to attachment of a taxane side chainprecursor to the 13-OH-position. In either of the schemes in U.S. Pat.No. 6,677,456, this means that the β-lactam intermediate is coupled tothe 13-OH-position of the completed pentacyclic taxane core. Thesemethods reduce the complexity and cost of synthesis of these toxiccompounds and result in a higher yield of final product. Accordingly,methods for the preparation of tesetaxel and other pentacyclic taxanesincorporating the novel compounds represented by formulas (Ia), (Ib),(III), (VII), (IX), (X) and (XI) are provided.

The method for synthesis of a taxane compound comprises coupling ataxane side chain precursor compound to the C13 hydroxyl group of thecompound represented by formula (Ia)

to produce a protected taxane reaction product, deprotecting theprotected taxane reaction product, and isolating the taxane compound.

In a specific example, a taxane side chain precursor compoundrepresented by formula (II):

wherein R² is an alkoxy group having from 1-6 carbon atoms or a halogenatom and R³³ is a protected hydroxyl group, is coupled to the C13hydroxyl of the compound represented by formula (Ia). A preferred R²substituent is fluorine at the 3-position of pyridine.

Compounds represented by formula (II) can be prepared by methods knownin the art as well as the inventive methods described herein. Forexample, where R³³ is triisopropylsilyl, the compound can be preparedusing the method described in Example 13 of U.S. Pat. No. 6,677,456 andin U.S. Pat. No. 7,126,003 B2.

Other pentacyclic taxanes according to the invention can be synthesizedby reacting a compound having a desired amino-containing group in placeof the dimethylaminomethyl group of the compound represented by formula(Ia) with a compound having a desired pyridine or pyridine derivative inplace of the fluoropyridine group of the compound represented by formula(II). In one such compound, the dimethylaminomethyl group of formula(Ia) is replaced by morpholinomethyl. For example, R² of formula (II)may be an alkoxy group having from 1 to 6 carbon atoms or an alternativehalogen atom such as chlorine.

The compound represented by formula (Ia) can be derived from either thecompound represented by formula (III):

or the compound represented by formula (IV):

using the relevant portions of Synthetic Method 1 or Synthetic Method 2,respectively, in U.S. Pat. No. 6,677, 456. These methods includeoxidation of the terminal double bond (i.e., the olefin) to remove onecarbon and produce an aldehyde. The aldehyde is reductively aminatedwith dimethylamine with hydrogenation as necessary.

The compound represented by formula (Ia) may be prepared by convertingthe terminal olefin group of the cyclic acetal of the compoundrepresented by formula (III) or formula (IV) to a diol group, forexample using an alkali metal permanganate or osmium tetroxide. The diolis oxidatively cleaved to an aldehyde (e.g., using periodate) andconverted to a dimethylaminomethyl group. These reactions are taught inconnection with different intermediate compounds in U.S. Pat. No.7,456,302 or U.S. Pat. No. 6,677,456, both of which are herebyincorporated by reference in their entirety. If the starting compoundfor this reaction is the compound represented by formula (IV), theproduct of the above reaction is the compound represented by formula(Ib) and the C6-C7 double bond is subsequently reduced to provide thecompound represented by formula (Ia).

The cyclic acetal ring of the compounds represented by formulas (III)and (IV) can be formed using the same or similar methodology asdescribed in connection with different intermediate compounds in U.S.Pat. No. 6,646,123, hereby incorporated by reference in its entirety.The method includes the use of acrolein dialkyl acetals (such asacrolein dimethyl acetal, acrolein diethyl acetal) with an acid catalyst(for example, camphorsulfonic acid) and, optionally, triethylamine, orwith a Lewis acid catalyst (for example, zinc chloride).

The invention provides an alternative method for synthesis of thepentacyclic core of tesetaxel (i.e., the compound represented by formulaIa) which is more economical and practical than methods of the priorart. It will also be appreciated by one of skill in the art that thesynthesis methods of the invention can be adapted to produce the corestructures of other pentacyclic taxanes. Included in the reactions ofthe invention are syntheses for the novel intermediates represented byformula (Ia), formula (Ib), formula (III), formula (VII), formula (IX),formula (X) and formula (XI).

The method of making a taxane compound according to the inventioncomprises coupling a taxane side chain precursor compound to theC13-hydroxyl of the compound represented by formula (Ia) to produce aprotected taxane reaction product, deprotecting the protected taxanereaction product, and isolating the taxane compound. A specific exampleof a reaction scheme for synthesis of the pentacyclic tesetaxel core(i.e., the compound represented by formula (Ia)) is illustrated in FIG.1.

Referring to FIG. 1, in a first aspect the compound represented byformula (Ia) can be synthesized according to Steps 1-7, starting with10-DAB III. In this embodiment, the compound represented by formula (Ia)is derived from the compound represented by formula (Ib), which isderived from the compound represented by formula (VII), which is derivedfrom the compound represented by formula (IX):

-   -   Step-1: Formylation of C10 of 10-DAB III. (e.g., using        Tf₂O/DMAP/DMF);    -   Step-2: Triflic anhydride reaction of the C7 hydroxyl. (e.g.,        using Tf₂O/Pyridine/CH₂Cl₂);    -   Step-3: Formation of a C6-7 double bond and hydrolysis of the        C10 formyl ester to produce the compound represented by formula        (IX). (e.g., a. Base such as Me₂NH/THF, b. Base such as        DBU/THF);    -   Step-4: Reduction of the C9 ketone to form a diol compound        having hydroxyls at C9 and C10, producing the compound        represented by formula (VII). (e.g., hydride reduction such as        BH₃, NaBH₄ or (Bu)₄NBH₄);    -   Step-5: Formation of a C9-C10 cyclic acetal attached to a        terminal olefin group, producing DHB, (acroline acetal and acid        catalyst (e.g., camphor sulfonic acid, TFA or TSA) or Lewis acid        (e.g., anhydrous zinc chloride));    -   Step-6: Oxidative cleavage of the terminal olefin group of the        cyclic acetal to form an aldehyde, and reductive amination of        the aldehyde, producing the compound represented by formula        (Ib). (a. conversion of olefin to diol, e.g., KMnO₄ or OsO₄, b.        oxidative cleavage of the diol to an aldehyde, e.g., periodate,        e.g., NaIO₄, c. conversion of the aldehyde to a        dimethylaminomethyl group, e.g., Me₂NH/AcONa/NaBH(OAc)₃); and    -   Step-7: Hydrogenation of the C6-7 double bond to produce the        compound represented by formula (Ia). (e.g., Rh—Al2O3/H2,        Pd—C/H₂ or Pd—C/HCOONH₄).

Again referring to FIG. 1, in an alternative route for synthesis of thecompound represented by formula (Ia), the reaction proceeds as describedabove from Steps 1-4. In this embodiment, the compound represented byformula (Ia) is derived from the compound represented by formula (III),which is derived from the compound represented by formula (VII), whichis derived from the compound represented by formula (IX). Following Step4, the synthesis proceeds as follows to produce the compound representedby formula (III):

-   -   Step-8: Hydrogenation of the C6-7 double bond of the diol        compound represented by formula (VII) to produce DOH. (e.g.,        Rh—Al₂O₃/H₂ or Pd—C/H₂ or Pd—C/HCOONH₄);    -   Step-9: Formation of a C9-C10 cyclic acetal attached to a        terminal olefin group, producing the compound represented by        formula (III). (e.g., acroline acetal/CSA (camphor sulfonic        acid) or other acid catalyst (for example, TFA or TSA or Lewis        acids such as anhydrous zinc chloride)).

The compound represented by formula III is then converted directly tothe compound represented by formula (Ia) by oxidative cleavage of theterminal olefin group to form an aldehyde and reductive amination of thealdehyde. (a. conversion of olefin to diol, e.g., KMnO₄ or OsO₄, b.oxidative cleavage of the diol to aldehyde, e.g., periodate, e.g.,NaIO₄, c. conversion of aldehyde to a dimethylaminomethyl group, e.g.,Me₂NH/AcONa/NaBH(OAc)₃) as shown in Step 11 of FIG. 1.

Further alternative syntheses provided by the invention for producingthe compound represented by formula (Ia) include alternative methods forderiving the compound represented by formula (IX) (the precursor of thecompound represented by formula (VII) from 10-DAB III. In a firstalternative reaction scheme illustrated as Step 10 in FIG. 1 1, and asStep 1 and Step 2 in FIG. 4, 10-DAB III is converted to the compoundrepresented by formula (IX) by formation of a C6-C7 double bond. Thedouble bond may be formed, for example, by reaction of the C7 hydroxylof 10-DAB III with triflic anhydride (e.g., Tf₂O/Pyridine/CH₂Cl₂)followed by base such as (DBU)/THF) to form the C6-C7 double bond of thecompound represented by formula (IX). The reaction then proceeds throughSteps 4-7 of FIG. 1 as described above to obtain the compoundrepresented by formula (Ia). Alternatively, the reaction steps canproceed through Step 4, Step 8, Step 9 and Step 11 of FIG. 1 aspreviously described.

Referring to FIG. 3, an alternative reaction scheme for synthesis of apentacyclic taxane is illustrated using tesetaxel as an example. Thissynthesis does not involve the C10 formyl ester intermediate of FIG. 1.In this alternative aspect of the invention the compound represented byformula (Ia) is derived from the compound represented by formula (III),which is derived from the compound represented by formula (IX). Thecompound represented by formula (Ia) can be synthesized according toSteps 1-8 of FIG. 3, starting with 10-DAB III:

-   -   Step 1: Reaction of the C7 hydroxyl of 10-DAB III with triflic        anhydride. (e.g., Tf₂O/Pyridine/CH₂C1 ₂);    -   Step 2: Formation of a C6-C7 double bond by base elimination to        form the compound represented by formula IX. (e.g., DBU);    -   Step 3: Reduction of the C6-7 double bond by hydrogenation of        compound IX to produce the compound represented by formula (XI).        (e.g., with Rh—Al₂O₃/H₂ or Pd—C/H₂ or Pd—C/HCOONH₄);    -   Step 4: Reduction of the C9 ketone to produce a diol compound        having hydroxyls at C9 and C10 (DOH). (e.g., using BH₃, NaBH₄ or        (Bu)₄NBH₄);    -   Step 5: Formation of a C9-C10 cyclic acetal from the diol DOH to        produce the compound represented by formula (III), wherein the        cyclic acetal is attached to a terminal olefin group. (acroline        acetal and acid catalyst (e.g., camphor sulfonic acid, TFA or        TSA) or Lewis acid (e.g., anhydrous zinc chloride)); and    -   Steps 6-8: Oxidative cleavage of the terminal olefin group to an        aldehyde and reductive amination of the aldehyde to produce the        compound represented by formula (Ia). (a. conversion of olefin        to diol, e.g., KMnO₄ or OsO₄, b. oxidative cleavage of the diol        to the aldehyde, e.g., periodate, e.g., NaIO₄, c. conversion of        aldehyde to a dimethylaminomethyl group, e.g.,        Me₂NH/AcONa/NaBH(OAc)₃). The reactions of Steps 6-8 can be        accomplished in a single operation without any purification of        intermediates.

Yet another alternative approach to synthesis of the compoundrepresented by formula (Ia) is illustrated in FIG. 4. This synthesisalso eliminates formation of the C10 formyl ester shown in FIG. 1. In afirst embodiment, the compound represented by formula (Ia) is derivedfrom the compound represented by formula (Ib), which is derived from thecompound represented by formula (IV), in a reaction scheme with earlyformation of the cyclic acetal. This first reaction scheme is shown inFIG. 4 as follows, starting with 10-DAB III:

-   -   Step 1: Reduction of the C9 ketone of 10-DAB III, producing a        triol compound (formula 10) having hydroxyls at C9 and C10        (e.g., using borohydride);    -   Step 2: Formation of a C9-C10 cyclic acetal from the triol        compound (formula 10), wherein the cyclic acetal is attached to        a terminal olefin group, (acroline acetal and acid catalyst        (e.g., camphor sulfonic acid, TFA or TSA) or Lewis acid (e.g.,        anhydrous zinc chloride)); and    -   Step 3: Triflic anhydride reaction of the C7 hydroxyl (e.g.,        Tf₂O/Pyridine/CH₂Cl₂), followed by base elimination to form a        C6-C7 double bond, producing the compound represented by formula        (IV);    -   Step 4: Oxidatively cleaving the terminal olefin group of the        cyclic acetal to an aldehyde, and reductively aminating the        aldehyde to produce the compound represented by formula (Ib).        (a. conversion of olefin to diol, e.g., KMnO₄ or OsO₄, b.        oxidative cleavage of diol to aldehyde, e.g., periodate, e.g.,        NaIO₄, c. conversion of aldehyde to a dimethylaminomethyl group,        e.g., Me₂NH/AcONa/NaBH(OAc)₃). These reactions can be        accomplished in a single operation without purification of any        intermediates to produce the compound represented by formula        (Ib);    -   Step 5: Hydrogenation of the C6-C7 double bond of the compound        represented by formula (Ib) to produce the compound represented        by formula (Ia). (e.g., Rh—Al₂O₃/H₂ or Pd—C/H₂ or Pd—C/HCOONH₄)

In an alternative embodiment shown in FIG. 4 to obtain the compoundrepresented by formula (IV), and subsequently the compound representedby formula (Ia), the compound represented by formula (IV) is derivedfrom the compound represented by formula (VII), which is derived fromthe compound represented by formula (X). That is, as further illustratedin FIG. 4, the triol compound 10 obtained by reduction of the C9 Ketonein Step 1 may be further reacted as follows:

-   -   Step 6: Triflic anhydride reaction of the C7 hydroxyl of the        triol compound 10 to produce the compound represented by formula        (X). (e.g., Tf₂O/Pyridine/CH₂Cl₂);    -   Step 7: Base elimination to form a C6-C7 double bond in the        compound represented by formula (X), producing the compound        represented by formula (VII);    -   Step 8: Formation of a C9-C10 cyclic acetal attached to a        terminal olefin group, producing the compound represented by        formula (IV). (acroline acetal and acid catalyst (e.g., camphor        sulfonic acid, TFA or TSA) or Lewis acid (e.g., anhydrous zinc        chloride)); and    -   Performing Step 4 and Step 5 of the first embodiment of the FIG.        4 reaction scheme as described above to obtain the compound        represented by formula (Ia).

The compound represented by formula (Ia), produced by any of theforegoing methods, can then be coupled at the C13 hydroxyl position to ataxane side chain precursor compound using any appropriate method knownin the art. For example, a side chain precursor compound according toformula (II) can be coupled to the compound represented by formula (Ia)to produce a variety of pentacyclic taxane final products

A specific example of such coupling using a β-lactam side chainprecursor to obtain tesetaxel is illustrated in FIG. 3, wherein aprotected β-lactam precursor (+)-THA is converted to (+)-TBA) (Step 9),coupled to the C13 hydroxyl group of the pentacyclic taxane corecompound represented by formula (Ia) using a hindered soluble alkalinemetal base, e.g., LHMDS (Step) 10, and the protecting group of the sidechain of the coupled product is deprotected (Step 11) using e.g., TBAF.The β-lactam precursor TBA(1-(tert-butoxycarbonyl)-4-(3-fluoro-2-pyridyl)-3-triisopropylsilyloxy-2-azetidinone)),is disclosed in U.S. Pat. No. 7,126,003 B2 and in the U.S. Pat. No.6,677,456 (Soga).

Alternatively, the side chain precursor may be a functional straightchain equivalent of the β-lactam such as TBBE (S-(4-Bromophenyl)(2R,3S)-3-[(tert-butoxycarbonyl)amino]-3-(3-fluoro-2-pyridinyl)-2-[(triisopropylsilyl)oxy]propanethioate), disclosed in U.S. Pat. No. 7,678,919 (Imura). Thecoupling reaction of the compound represented by formula (Ia) with TBBEis illustrated in FIG. 5 and discussed in more detail below.

Conventional synthesis of β-lactams for use as side chains precursorsfor coupling to taxane core compounds may use p-anisidine to make theacetoxyphenylazetidine (APA) intermediate, which necessitates the use ofceric ammonium nitrate (CAN) at a later step to remove the 1-phenylsubstituent and convert TPA to THA. See, for example, U.S. Pat. No.5,336,785 (Holton). This is an impractical chemistry for use on acommercial scale due to poor yield, quality issues and significant wastegeneration.

In an additional embodiment that addresses these problems, the inventionprovides a method for synthesis of the β-lactam side chain precursor foruse in taxane synthesis which employs methoxy methylethoxy (MOP, ormethoxydimethyl propyloxy) or other acetal groups for protection of the3-OH of the β-lactam side chain precursor as disclosed in U.S. Pat. No.6,310,201 (Thottathil), which is incorporated by reference herein.Although this chemistry has generally been described in U.S. Pat. No.7,176,326 (Thottathil) and U.S. Pat. No. 6,310,201, it has notpreviously been applied to a β-lactam wherein the 4-substituent isheterocyclic and halogen-substituted as required for the synthesis oftesetaxel. The halogen substituent on the side chain, particularly thefluorine substitution of taxanes such as tesetaxel, is of particularinterest and utility as it allows PET imaging of taxane distribution inthe body, especially to cancer sites and cancer cells. The fluorinatedpyridine is also particularly important for the enhanced biologicalactivity of tesetaxel as a chemotherapeutic agent.

In this aspect the invention provides taxane side chain precursorcompounds represented by formula (V) and formula (VI):

wherein Me is methyl and BOC is tert-butoxycarbonyl. The compoundsrepresented by formulas (V) and (VI) may be synthesized generally asdescribed in U.S. Pat. No. 7,176,326 and U.S. Pat. No. 6,310,201. Itwill also be appreciated by those skilled in the art that other groupsfor protection of the 3-OH of the β-lactam side chain precursor asdisclosed in U.S. Pat. 6,310,201 (Thottathil) may be substituted for MOPin formula (V) and formula (VI).

However, in another aspect the invention provides an alternativesynthesis that avoids the use of CAN while producing a crystalline solidβ-lactam side chain precursor for linkage to C13 of the taxanepolycyclic core structure. The synthesis is performed according to thegeneral reaction scheme illustrated in FIG. 2, wherein Py is pyridine orsubstituted pyridine; Ac is acetyl; Me is methyl; MOP is2-methoxypropyl; and BOC is tert-butoxycarbonyl. It will also beappreciated by those skilled in the art that other groups for protectionof the 3-OH of the β-lactam side chain precursor as disclosed in U.S.Pat. No. 6,310,201 (Thottathil) may be substituted for MOP in theforegoing reaction scheme. In a particular embodiment Py is ahalo-substituted pyridine, such as fluoropyridine, or amethoxy-substituted pyridine. In a preferred embodiment Py is3-fluoropyridine. When Py is 3-fluoropyridine, compound 18 of the abovereaction scheme is the compound represented by formula (VI) and compound17 is the compound represented by formula (V). Py is also intended toencompass aromatic substituents and other suitable heteroaromaticmoieties.

The invention also provides a compound represented by formula (VIII)which is a novel intermediate in the reaction scheme of FIG. 2 forsynthesis of the compounds represented by formula (V) and formula (VI).

wherein Py is as defined above.

The above reaction proceeds by reacting ammonia with the aldehyde PyCHOto produce the compound represented by formula (VIII). Treatment of thecompound represented by formula (VIII) with acetoxy acetyl chloride, TEAand THF forms the β-lactam ring (Compound 19a in FIG. 2) and the1-substituent is removed using a mixture of hydrochloric acid in water(hydrolysis) or a mixture of Pd—C (palladium carbon) and hydrogen(reduction) to form Compound 20. Compound 20a is formed by enzymaticresolution of Compound 20 enantiomers generally as described in U.S.Pat. No. 7,176,326. In this process Compound 20 is treated with lipase,pen-amidase or esterase and the desired enantiomer is recovered byrecrystallization. Base hydrolysis of Compound 20a (for example usingK₂CO₃) removes the acetyl to produce the 3-OH (Compound 21), which isthen protected by addition of methoxypropene and pyridinium p-toluenesulfonate (PPTS), generally ay described in U.S. Pat. No. 6,130,201,resulting in Compound 17. Alternatively, compound 17 is prepared byaddition of methoxypropene/CSA or 2,2-dimethoxypropane/CSA. BOC is addedto Compound 17 by addition of (BOC)₂O/DMAP to form the final productCompound 18.

In a specific embodiment for synthesis of the β-lactam side chainprecursor for use in preparing tesetaxel, the aldehyde starting compoundis a derivative of 3-fluoropyridine and the reaction scheme is asillustrated in FIG. 2.

For preparation of the tesetaxel side chain precursor as shown in FIG.2, the 2-aldehyde of FFP (3-fluoropyridine) is reacted with ammonia(Step 1). Subsequent steps to prepare the compound represented byformula (VI) are as described above. In a preferred embodiment thecompound represented by formula (V) is prepared using methoxypropene/CSAor 2,2-dimethoxypropane/CSA and the BOC group is added using(BOC₂)O/DMAP.

The compound represented by formula (VI) is coupled to the C13 hydroxylof the compound represented by formula (Ia) to produce protectedtesetaxel or another related pentacyclic taxane compound. The side chainlinking reaction is preferably accomplished using a hindered solublealkaline metal base such as lithium hexamethyl disilazide (LHMDS), whichhas been described in US Patent Publication 2002/0091274 (Holton), U.S.Pat. No. 6,794,523 (Holton) and U.S. Pat. No. 6,350,887 (Thottathil) forlinkage of side chains to the C13 hydroxyl of 7-protected taxanes.However, other metallic bases may also be used for coupling of taxaneside chains as disclosed in U.S. Pat. No. 6,350,887. Alternatively,attachment of the of the β-lactam intermediate represented by formula(VI) to the 13-position of the pentacyclic taxane intermediaterepresented by formula (Ia) can be performed as described in Example 6in U.S. Pat. No. 6,677,456.

The reaction scheme using a soluble hindered base for coupling theβ′-lactam side chain precursor is as follows:

wherein HTX refers to the intermediate compound in which the 2′ hydroxylis protected by R and R is as indicated in the reaction scheme above.The compound represented by formula (Ia) is first reacted with LHMDS oranother suitable alkaline metal base in a solvent such astetrahydrofuran (THF) as taught in Examples 7 and 9 of U.S. Pat. No.6,677,456. Upon addition of the selected β-lactam to the reactionmixture, the 13-position OH reacts with the β-lactam to produce HTX. The2′-OH of HTX is deprotected by treatment with mild acid or TBAF(tetrabutylammonium fluoride) generally as taught in Examples 7 and 9 inU.S. Pat. No. 6,677,456. The tesetaxel final product is purified and,optionally, crystallized to obtain the desired polymorph.

Alternatively, the β-lactam intermediate represented by formula (VI) canbe converted to the functional equivalent TBBE as described below withrespect to FIG. 5, and coupled to the taxane core compound representedby formula (Ia).

An example of a reaction scheme for obtaining TBBE and coupling the TBBEside chain precursor is shown in FIG. 5. As shown, TBA is synthesized byconversion of4-(3-fluoro-2-pyridyl)-3-triisopropylsilyloxy-2-azetidinone (THA) to TBAby reaction with a butoxycarbonyl group. The 3-hydroxyl of the THA andTBA precursors can be protected by any hydroxyl protecting group, suchas triisopropylsilylether (TIPS). TBBE can be derived from TBA bythio-esterification of TBA (the compound represented by formula (II))with a thiol compound such as 4-bromothiophenol or 4-bromobenzenethiolin the presence of a base. This process is described in U.S. Pat. No.7,678,919 (Imura). TBBE is then coupled to C13 hydroxyl group of thepentacyclic taxane core compound (e.g., the compound represented byformula (Ia)) in an inert solvent in the presence of base to produce ataxane with a hydroxyl-protected side chain. Coupling may be mediated byeither bases such as sodium hydride or by soluble hindered bases such asLHMDS, and is preferably carried out in an inert gas atmosphere, such asnitrogen or argon. The coupled, protected product (9 in FIG. 3 and FIG.5) is isolated and purified, and the side chain is deprotected toproduce the final taxane compound. The final product may optionally becrystallized to obtain the desired polymorph.

One method for crystallization of tesetaxel is described in U.S. Pat.No. 7,410,980 (Uchida). This method uses acetone, a mixture of acetoneand water, or a mixture of acetonitrile and water for crystallization;however, other methods for purification of tesetaxel by crystallizationmay be employed. Other solvents such as ethanol, methanol, isopropanol(each with or without water) may also be used for crystallization as isknown in the art.

If necessary, steps may be taken to control and minimize hydrolysis ofthe BOC group of HTX by the acid deprotection reaction. For example,reducing the reaction temperature, shortening the reaction time andvarying the reaction conditions may be employed to minimize hydrolysisof the BOC group if necessary. Alternatively, in the event of anundesirable amount of BOC hydrolysis the BOC group may simply bere-added by reaction of HTX with Boc₂O in DMAP as described above.

Alternatively to coupling to the compound represented by formula (Ia) toproduce 2′-O-protected tesetaxel (HTX), the compound represented byformula (II) or formula (VI) can be coupled to the C13 hydroxyl of anyof the compounds represented by formula (Ib), formula (III) or formula(IV) using an alkaline metal base as described above to producealternative intermediates in the tesetaxel synthesis schemes describedabove. The coupled, protected product is then deprotected and purifiedas described above. Coupling the side chain precursor to the alternativeintermediates (represented by formula (III), formula (IV) and formula(Ib)) means that the side chain is added to the taxane core structurebefore completion of the tesetaxel core. These alternative intermediatesmay themselves be useful pentacyclic taxane compounds, but may also bereacted as described herein to complete the synthesis of the pentacyclictesetaxel core, i.e., removal of the C6-7 double bond from the compoundrepresented by formula (Ib); removal of one carbon from the terminalolefin of the cyclic acetal and addition of the dimethylaminomethylsubstituent to the compound represented by formula (III); or removal ofone carbon from the terminal olefin, addition of the dimethylaminomethylgroup, and removal of the C6-7 double bond for the compound representedby formula (IV).

The final tesetaxel product may also be converted to variouspharmaceutically acceptable salt forms using methods well known in theart. These salt forms will provide a variety of useful physico-chemicaland pharmacological properties to tesetaxel which will be useful indifferent medical applications. For example, acid addition salts oftesetaxel may be prepared through dissolution thereof in an appropriatesolvent in the presence of an appropriate acid prior to purificationand/or crystallization. The salt forms of tesetaxel may comprise onlytesetaxel and the acid, or they may be hydrates and/or solvates of theacid addition salt of tesetaxel. These acid addition salts of tesetaxelare represented by the following structural formula:

(TT)_(m).(HX)_(n).(H₂O)_(y).(Sol)_(z)   (formula (XII))

-   -   wherein    -   TT is tesetaxel;    -   HX is a monobasic, dibasic, tribasic or polybasic acid;    -   Sol is an organic solvent;    -   m and n are each independently an integer from about 1 to about        5; and    -   y and z are each independently an integer from 0 to about 5.

The salt forms of tesetaxel which are not hydrates and/or solvates mayhave the general structure (TT)_(m).(HX)_(n) wherein TT is tesetaxel, HXis an acid, and m and n are each independently an integer from about 1to about 5. That is, in these compounds both y and z of the compoundrepresented by formula (XII) are 0.

The salts of tesetaxel and a monobasic acid may be represented byformula (XII), wherein both m and n are 1, or wherein m is 1 and n is 2.These compounds may also be designated TT.HX or TT.2HX, respectively.The useful monobasic acids for forming salts of tesetaxel having thesestructures include HCl (hydrochloric acid), HBr (hydrobromic acid), HI(hydroiodic acid), HNO₃ (nitric acid), HOAc (acetic acid), benzoic acid,toluic acid (ortho, meta, para), lactic acid (both D and L), MSA(methane sulphonic acid), BSA (benzene sulphonic acid), esylate (ethanesulphonic acid), sulfuric acid, CSA (camphor sulphonic acid), TSA(toluene sulphonic acid ortho, meta, para), (S)-(+)-mandelic acid,(R)-(−)-mandelic acid, gentisic acid, hippuric acid, glycolic acid2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, gluconic acid,natural mono-basic amino acids and other pharmaceutically acceptableacids.

The salts of tesetaxel and a dibasic acid may be represented bystructural formula (XII), wherein m and n are both 1, or wherein m is 2and n is 1. These compounds may also be designated TT.HX or 2TT.HX,respectively. The useful dibasic acids for forming salts of tesetaxelhaving the TT.HX or 2TT.HX structure include malic acid, maleic acid,fumaric acid, oxalic acid, succinic acid, tartaric acid, malonic acid,adipic acid, itaconic acid, cyclohexane dicarboxylic acid (1,2; 1,3;1,4; both cis and trans), phthalic acid (1,2; 1,3; and 1,4,), edisylate(1,2-ethanedisulfonate), phenyl phosphonic acid, digluconic acid,natural di-basic amino acids, and other pharmaceutically acceptableacids.

The salts of tesetaxel and a dibasic acid may be represented bystructural formula (XII), wherein m is 1 and n is 2. These compounds mayalso be designated TT.2HX. Useful dibasic acids for forming salts oftesetaxel having the TT.2HX structure include malic acid, maleic acid,fumaric acid, oxalic acid, succinic acid, tartaric acid, malonic acid,adipic acid, itaconic acid, cyclohexane dicarboxylic acid (1,2; 1,3;1,4; both cis and trans), phthalic acid (1,2; 1,3; and 1,4, edisylate(1,2 ethanedisulfonate), phenyl phosphonic acid, digluconic acid,natural dibasic amino acids, and other pharmaceutically acceptabledibasic acids.

The salts of tesetaxel and tribasic or polybasic acids may berepresented by structural formula (XII), wherein m and n are eachindependently an integer from about 1 to about 5. Useful tribasic andpolybasic acids for forming salts of tesetaxel include citric acid,phosphoric acid, and other pharmaceutically acceptable tribasic andpolybasic acids.

The salts of tesetaxel and a monobasic, dibasic, tribasic or polybasicacid can also exist as a hydrate or solvate, or a combination of hydrateand solvate, which may be designated generally as(TT)_(m).(HX)_(n).(H₂O)_(y).(Sol)_(z), wherein either or both of y and zare not 0. That is, hydrates and solvates of tesetaxel are representedby structural formula (XII) wherein y and z are each independentlyintegers from about 1 to about 5, y is 0 and z is an integer from about1 to about 5, or z is 0 and y is an integer from about 3 to about 5.

In specific embodiments, hydrates and solvates of salts of tesetaxel anda monobasic acid may have the structure (TT).(HX).(H₂O)_(y).(Sol)_(z)(i.e., m and n are both 1) or (TT).2(HX).(H₂O)_(y).(Sol)_(z) (i.e., m is1and n is 2). Similarly, in specific embodiments of hydrates andsolvates of salts of tesetaxel and a dibasic acid the compounds may havethe structure (TT).(HX).(H₂O)_(y).(Sol)_(z) (i.e., m and n are both 1),2(TT).(HX).(H₂O)_(y).(Sol)_(z) (i.e., m is 2 and n is 1) or(TT).2(HX).(H₂O)_(y).(Sol)_(z) (i.e., m is 1 and n is 2). Hydrates andsolvates of salts of tesetaxel and tribasic or polybasic acids are morevariable in structure, but typically will be represented by formula(XII) wherein wherein m and n are each independently an integer fromabout 1 to about 5, and; y and z are each independently integers fromabout 1 to about 5, y is 0 and z is an integer from about 1 to about 5,or z is 0 and y is an integer from about 1 to about 5.

Appropriate organic solvents for formation of the solvate includeacetone, methanol, ethanol, propanol, butanol, acetonitrile,tetrahydrofuran, isopropyl alcohol, toluene, N,N-dimethylformamide, andother pharmaceutically acceptable solvents.

The foregoing acid addition salts of tesetaxel, including hydratesand/or solvates thereof, may be used in methods for treating cancer. Insuch methods the acid addition salt is administered to a cancer patient(human or other mammal) in an amount effective to treat the cancer.Administration may be by any appropriate route, including parenteraladministration, intra-cavitary administration, oral administration,injection, or infusion. Dosing is typically at or near the maximumtolerated dose in order to increase the response rate. To facilitateadministration for the treatment of cancer, the acid addition salt oftesetaxel will typically be formulated as a pharmaceutical preparationby addition of appropriate pharmaceutically acceptable excipients, whichmay include solubilizers, stabilizers, and the like. The form of thepharmaceutical preparation is considered in the choice of excipients.For example, the pharmaceutical preparation may be in the form of apowder, tablet, solution, emulsion or capsule designed for the desiredroute of administration. Specific examples of such pharmaceuticalpreparations include formulations for oral administration, extendedrelease, parenteral administration and intra-cavitary administration.

In a first aspect of the process of the invention for producing acidsalts of tesetaxel, the monobasic, dibasic, tribasic or polybasic acidselected for crystallization (in a suitable solvent) is added to thereaction mixture for linking the taxane side chain precursor to thecompound represented by formula (Ia), followed by deprotection of the2′-O group. The final acid salt product is then subsequently purifiedand crystallized from the crude reaction mixture as illustrated in thefollowing reaction scheme:

In an alternative embodiment, the monobasic, dibasic, tribasic orpolybasic acid selected for crystallization is added to a solution ofpurified and isolated tesetaxel, and the acid salt form is crystallizedfrom the mixture. The acid for crystallization may be in solution in asuitable solvent which is added to the purified and isolated tesetaxel(also dissolved in a solvent) followed by crystallization of the saltform. This process is illustrated in the following reaction scheme:

EXAMPLES Example 1 Coupling of Intermediate Compound (Ia) with the SideChain TBA. Procedure 1

A solution of the 13-hydroxy taxane intermediate (Ia) (715 mg) infreshly dried and distilled THF was prepared and cooled to −50° C. Thecooling was applied only after complete dissolution of the material. Tothis cooled solution was added drop wise with vigorous stirring lithiumhexamethyl disilazide (LHMDS, 1.05 eq. in THF; titrated with1,3-diphenyl acetone p-tosylhydrazone) over a period of several minutesto keep the internal temperature around −50° C. After the addition, thereaction mixture was warmed to −30° C. and stirred at that temperaturefor 5 minutes. A freshly prepared solution of TBA (1.1 eq.) in THF wasadded drop wise to the reaction mixture over a period of severalminutes. No significant exotherm was observed. The flask containing TBAwas washed with a few ml of THF and the washings were transferred to thereaction mixture. The resulting solution was brought to 0° C. byreplacing the cooling bath with an ice-water bath. The reaction wasstirred for an additional 90 minutes. The reaction was monitored by TLCand HPLC which indicated complete conversion to 2′-TIPS tesetaxel(intermediate 9).

The reaction was quenched with pH 7 phosphate buffer followed bysaturated aqueous NaHCO₃. It was then diluted with ethyl acetatefollowed by conventional extractive work-up and solvent concentration togive the coupled product in quantitative yield. The crude product wasfurther purified by silica gel polish filtration and 1.2 g of coupledmaterial was obtained. MS MW 1037. HPLC retention time (RT) 11.2minutes.

Example 2 Coupling of Intermediate Compound (Ia) with the Side ChainTBA. Procedure 2

In an alternate method, the procedure was repeated using 58 mg of pure13-hydroxy taxane core intermediate (Ia) as in Example 1. The yield ofthe reaction was close to 100% in 94% purity (yield 77 mg). MS MW 1037.HPLC retention time (RT) 11.2 minutes.

Example 3 Deprotection of 2′TIPS Group, Intermediate 9 to Tesetaxel

A solution of 2′ TIPS tesetaxel (intermediate 9) (77 mg) in ethanol-THF(1:1) solvent (5 ml) was cooled to 0° C. using an ice-bath. To thissolution pre-cooled (0° C.) 1.5 N HCl (aqueous) (1 ml) was added dropwise with vigorous stirring over a period of several minutes. Thereaction was stirred for several hours until HPLC and TLC indicatedcomplete disappearance of the starting 2′TIPS tesetaxel and the presenceof tesetaxel.

The conventional extractive work-up using ethyl acetate followed bysolvent evaporation gave tesetaxel in quantitative yield. Depending onthe purity, the crude product may be further purified by chromatographyand/or crystallization. Crude yield was 70 mg, 87% HPLC purity, HPLC RT7.9. Chromatographic purification gave 45 mg tesetaxel in 98% purity. MSMW 882.

Example 4 Alternate Deprotection of Intermediate 9 to Tesetaxel. TheTBAF Method

A solution of 2′ TIPS tesetaxel (intermediate 9) (1.2 gm) in ethylacetate (10 ml) was cooled to 0-10° C. and 1.1 equivalent oftetrabutylammonium fluoride solution was added and stirred for 0.5-3 h.Completion of the reaction was monitored by HPLC. This reaction wasconsidered completed when the starting material was ≦0.1%. At that time,4% sodium hydrogen carbonate and saturated saline were added, and theorganic layer was separated and washed with saturated saline, thenconcentrated under reduced pressure at ≦50° C. The crude tesetaxel atthis point can be purified either by chromatography or bycrystallization or by a combination of chromatography andcrystallization. The yield was 535 mg with HPLC purity of 95%. HPLC RT7.9., MS MW 882.

Example 5 Tesetaxel Crystallization

Crude tesetaxel was dissolved in acetone, activated carbon was added,and the mixture was stirred at 15-50° C. for 0.5-2 h. Insoluble matterwas filtered off with a micro filter (0.2-0.25 μm) and washed withacetone. Ultra filtered (UF) water at 40-50° C. was added, and thesolution was stirred for ≧6 h. The precipitate was isolated and washedwith cold 40% aqueous acetone. The precipitate was then dried underreduced pressure at ≦60° C.

For a second crystallization to increase the purity and/or to controlthe morphology, the crystals were dissolved in acetone at ≦50° C., waterwas added to effect crystallization, and the mixture was stirred at roomtemperature for ≧6 h. Precipitated crystals were collected by filtrationand washed with 40% aqueous acetone. The product was weighed and theyield of tesetaxel was calculated. The yield from crude tesetaxelobtained from crude 2′TIPS tesetaxel can be 60-80%. HPLC RT 7.9., MS MW882. The tesetaxel was dispensed into brown bottles.

Synthesis of tesetaxel as described in Examples 1-5 was confirmed byHPLC, as shown in FIG. 6.

Example 6 Preparation of TBA

0.9 moles of (+)-THA and 10 v/w (to THA) of THF were placed in areactor, dissolved to a solution while stirring, followed by addition of0.0361 w/w (to THA) of DMAP and 1.0 moles of (Boc)₂O. The reaction wasallowed to proceed at room temperature for 30 to 90 min. Completion ofthe reaction was checked by HPLC. Upon completion, 5 v/w of a 4% sodiumbicarbonate solution was added and extraction was performed using 10 v/w(to THA) of n-hexane. The organic layer was washed with about 5 v/w tapwater, and then dried over an appropriate amount of magnesium sulfate.Insoluble substances were removed by filtration and washed with about 2v/w (to THA) of n-hexane. The filtrate, combined with the washings, wasconcentrated under reduced pressure at 40° C. or less to obtain TBA as aresidue. HPLC Retention Time (RT) is 17.4 minutes, MS MW 439. Yield100%. This residue was used for ail tesetaxel coupling experiments.

Example 7 Preparation of TBBE

Crude TBA and 1.1 equivalent of 4-BTP (4-bromomo-thiophenol) weredissolved in 13 v/w of IPE (isopropyl ether), 0.3 equivalent ofpotassium carbonate was added, and the mixture was stirred at roomtemperature for 0.5 to 3 hours. Completion of the reaction was checkedby HPLC.

Upon completion seven volumes of IPE and nine volumes of tap water wereadded and the organic layer was separated. The organic layer was washedwith nine volumes of saturated saline, and then dried over anappropriate amount magnesium sulfate. Insoluble substances were removedby filtration and washed with 2 volumes of IPE. The filtrate combinedwith washings was concentrated under reduced pressure at 40° C. or lessto obtain TBBE as a residue. Yield quantitative. HPLC purity 97%, RT21.8 minutes. MS MW 628. This residue was used for all tesetaxelcoupling experiments.

Example 8 Coupling of Intermediate Compound (Ia) with the Side ChainTBBE. Procedure 1

3.0 Equivalent of NaH and 8 volumes of DME (compared to 13-hydroxytaxane core to be used) were placed in a reactor and stirred. 0.2 to 5 gof 13-hydroxy taxane core Ia dissolved in 7 volumes of dry DME wasadded, followed by 1.1 equivalent of crude residue TBBE dissolved in 5volumes of dry DME at an internal temperature of 10° C. or less. Coolingwas stopped, and the reaction was allowed to proceed for 1 to 4 hours.Completion of the reaction was checked by HPLC. Upon completion, thereaction was quenched with a mixture of 9 volumes of 4% sodiumbicarbonate solution and 9 volumes of ethyl acetate, and the organiclayer was separated. The organic layer was washed with a mixture of 9volumes of tap water and 6 volumes of saturated saline, and concentratedunder reduced pressure at 50° C. or less. The residue obtained is 2′TIPS protected tesetaxel (compound 9).

The 2′TIPS protected tesetaxel was further purified by crystallizationor chromatography and/or a combination of both chromatography andcrystallization. MS MW 1037. HPLC Retention time (RT) 11.2 minutes.

The crude product obtained was also used as-is for the next deprotectionstep to tesetaxel.

Example 9 Alternate Coupling of Intermediate Compound (Ia) with the SideChain TBBE. Procedure 2

The 13-hydroxy taxane core (compound Ia) (0.2 to 5 g) and 8 volumes ofdry THF were placed in a reactor, cooled to −50° C. and stirred. 1.1equivalents of LHMDS in THF (1 M) was added to the reaction and themixture was stirred for 20 minutes at −50-30° C. 1.1 equivalents ofcrude residue TBBE dissolved in 5 volumes of dry THF was added and theinternal temperature was raised to 0-10° C. Cooling was stopped, and thereaction was allowed to proceed for 1 to 4 hours. Completion of thereaction was checked by HPLC. Upon completion the reaction was quenchedwith a mixture of 9 volumes of 4% sodium bicarbonate solution and 9volumes of ethyl acetate, and the organic layer was separated. Theorganic layer was washed with a mixture of 9 volumes of tap water and 6volumes of saturated saline, and concentrated under reduced pressure at50° C. or less. The residue obtained is 2′ TIPS protected tesetaxel(compound 9).

The 2′ TIPS protected tesetaxel was further purified by crystallizationor chromatography and/or a combination of both chromatography andcrystallization. MS MW 1037. HPLC Retention time (RT) 11.2 minutes.

The crude product obtained was also used as is for the next deprotectionstep to tesetaxel.

Example 10 Triflic Anhydride Reaction; Conversion of 10-DAB III toIntermediate 2 of FIG. 3

10 ml of pyridine, 2.9 g of 10-DAB III, and 2.10 g of4-dimethylaminopyridine (DMAP) were added to the reactor. The reactionmixture was chilled and maintained under controlled temperature between0° C. and 10° C. under nitrogen atmosphere. 1.94 g oftrifluoromethansulfonic acid/anhydride was added drop wise over a periodof several minutes. During the addition the reaction mixture wasmaintained between 0° C. and 10° C. The reaction mixture was checked byHPLC for completion. Conventional extractive work-up gave the crudeproduct as a mixture of the triflate (intermediate 2 in FIG. 4) and thecorresponding eliminated 6-7 olefin (compound IX). The crude materialwas used as-is for the next step. MS MW 677, HPLC retention time (RT)9.7 minutes.

Example 11 Alternate Procedure for 7-Hydroxy Triflation; GeneralProtocol

A solution of 10-deacetyl baccatin (10-DAB, 1 equivalent) and pyridine(2.9 volumes, 20 equivalents) was stirred in CH₂Cl₂ (2 volumes) andcooled to −20° C. under nitrogen atmosphere. Trifluoromethanesulfonicanhydride (OTf₂) in CH₂Cl₂ solution (2 volumes) was added over 4 hours,keeping the internal temperature at 0° C. under nitrogen atmosphere. Theresulting mixture was stirred and monitored by TLC. The reaction mixturewas quenched by addition of THF (10 volumes) and HCl (1 N; 6 volumes)then the THF layer was washed with NaHCO₃ and NaCl saturated solutions.Evaporation of the solvent gave the crude triflate. Purification wasrealized by washes with DCM/MeOH 98:2.

20 g of 10-DAB gave 14 g of triflate intermediate (intermediate 2 ofFIG. 3) with a chemical purity of 84% and a yield of 70%. MS MW 677.HPLC Retention time (RT) 9.7 minutes.

Example 12 Elimination of the 7-Triflate (Intermediate 2 of FIG. 3) to6-7 Olefin (Compound IX). General Protocol

A solution of 7-OTf-10DAB (intermediate 2 of FIG. 3) (1 equivalent) andDBU (5 equivalents) was stirred in THF (6.2 volumes). The resultingmixture was stirred at reflux (70° C.) for 2 h and monitored by HPLC.The reaction mixture was quenched by addition of EtOAc (10 volumes). Thesolution was washed with saturated NH₄Cl and saturated aqueous NaCl. Theorganic layer was dried (MgSO₄), filtered and evaporated to dryness. Thecrude compound was purified by flash chromatography (Merck 40-63 μm)with DCM/MeOH 98:2. 14 gm of triflate (intermediate 2 of FIG. 3) gave 9g of desired compound (compound IX) as a white powder, aftercrystallization in DCM., HPLC chemical purity is 95%. The yield is 80%.MS MW 527. HPLC Retention time (RT) 4.9 minutes.

Example 13 Reduction of C6-7 Double Bond: Conversion of Compound (IX) toIntermediate XI of FIG. 3

The C6-7 olefin of compound (IX) obtained above was dissolved in 5volumes of ethanol and 0.5 volume of water was added. 10% Pd/C 50% wet(5% wt) and ammonium formate (2×5 equivalents) was added and stirredunder nitrogen atmosphere at 40 to 60° C. for 1 to 4 hours. Completionof the reaction was checked by HPLC.

Insoluble substances were removed. The residue was washed with 3 volumesof ethanol, then concentrated under reduced pressure at 50° C. or less.To the concentrated residue, 15 volumes of ethyl acetate and 3 volume of4% sodium hydrogen carbonate and 3 volume of saturate saline were added,and the organic layer was separated. The organic layer was washed with 7volumes of saturated saline, then dried over an appropriate amount ofmagnesium sulfate. Insoluble substances were removed, and the residuewas washed with 3 volumes of ethyl acetate and again concentrated underreduced pressure at 50° C. or less. The crude product was purified bychromatography and/or crystallization to obtain intermediate XI.

HPLC chemical purity was 67%. The yield was 95%. MS MW 525. HPLCRetention time (RT) 10.7 minutes.

Example 14 Borane Reduction of Intermediate XI to DOH

The 9-carbonyl group of intermediate XI was reduced to the correspondingbeta alcohol by the reducing agent borane-THF complex. Intermediate XI(700 mg) was dissolved in THF (10 ml) and was cooled to −10° C. undernitrogen atmosphere. 15 hydrogen equivalents of borane-THF was addeddrop wise and the temperature was brought to 0° C. After stirring thereaction for 2 hours, an additional amount of borane-THF (5 equivalents)was added to the reaction. After stirring the reaction for another twohours, it was quenched by adding the reaction mixture into ice-watercontaining 0.1% formic acid. Extractive work-up followed bychromatography gave 77% yield of the DOH product.

HPLC chemical purity was 77%. The yield was 90%. MS MW 531. HPLCretention time (RT) 10.0 minutes.

Example 15 Preparation of Acetal (Conversion of Intermediate 10 toIntermediate 13 of FIG. 4)

35 L of AcOMe, 3.68 kg of alcohol intermediate 10 (FIG. 4), 0.46 kg oftriethylamine HCl salt (TEA.HCl), and 2.63 kg of acrolein diethyl acetal(ADA) were added to the reactor. 14.1 g of camphorsulfonic acid (CSA)was dissolved to 1.8 L of AcOEt, the solution was added to the reactionmixture, and the temperature was kept between 15° C. and 25° C. forseveral hours (from 4 hours to 28 hours). The reaction mixture waschecked by HPLC for completion. 37 L of isopropyl ether (IPE) was added,and 29 L of n-hexane was added. The mixture was chilled to between 10°C. and 0° C. and stirred for from 1 to 3 hours while maintaining thetemperature. After stirring, the precipitate was obtained by filtrationusing a 60 cm Nutsche filter. The precipitate was washed with 15 L ofIPE. The precipitate was then dissolved in 74 L of AcOEt. The organiclayer was washed with 37 L of water followed by 18 L of water (twice).Next, 9 L of 4% NaHCO₃ and 9 L of saturated NaCl solution were mixed andused to wash the organic layer. The organic layer was then dried with 2kg of MgSO₄. It was filtered and the residue was washed with 18 L ofAcOEt. The combined organic layer was evaporated to a residue undercontrolled temperature between 20° C. and 40° C. 22 L of IPE was addedto the residue, and the organic layer was stirred under controlledtemperature between 20° C. and 30° C. 22 L of n-hexane was added to themixture, and the mixture was stirred not less than 1 hour. Afterchilling under controlled temperature between 10° C. and 0° C., themixture was stirred not less than 1 hour. The precipitate was filteredwith a 60 cm Nutsche (SUS) filter and washed with 11 L of IPE. Afterdrying with a vacuum drier, temperature controlled between 20° C. and40° C., the crystalline form was obtained. (Standard 2.01 kg (Yield51%), Theoretical 3.94 kg, Specification; not less than 80% by HPLC),

Example 16 Preparation of Acetal Compound (III): Procedure 1

The diol compound DOH (1 g) was dissolved in dichloromethane (10 ml) and4 equivalents of acrolein dimethyl acetal was added to the reactionmixture. Powdered anhydrous zinc chloride (0.2 equivalents) was addedand the reaction mixture was stirred at about 30° C. for 24 hours untilHPLC analysis indicated complete reaction. Extractive work-up followedby chromatographic purification gave 82% yield in 96% purity. MS MW 568.HPLC Retention time (RT) 13.9

Example 17 Alternate Procedure for the Preparation of Acetal Compound(III): Procedure 2

The same procedure as that used for the conversion of intermediate 10 tointermediate 13 in Example 15 above was applied for the conversion ofDOH to acetal compound (III) in 85% yield and 95% purity. MS MW 568.Retention time (RT) 13.9

Example 18 Conversion of Compound (III) to Compound (Ia)

0.17 to 7.9 g of the acetal compound III and 15 v/w of pyridine wereplaced in a reactor and dissolved to a solution, followed by addition of2.5 v/w of tap water. The internal temperature was maintained between25° C. and 55° C. 4.09 v/w of a potassium permanganate solution (50 gper liter of water) was added, and the reaction was allowed to proceedfor 0.3 to 3 hours. The residual amount of acetal compound (III) waschecked by HPLC. 15 v/w of ethyl acetate, 5 v/w of a 10% aqueous citricacid solution and 3 v/w of saturated saline were added, and the organiclayer was separated. The organic layer was washed with a mixture of 3v/w of a 10% aqueous citric acid solution and 3 v/w of saturated saline,followed by a wash with a mixture of 5 v/w of 4% sodium bicarbonatesolution and 3 v/w of saturated saline. The washed organic layer wasthen concentrated under reduced pressure at 50° C. or less to obtain thediol intermediate as a residue.

The diol intermediate residue was dissolved in 7 v/w of acetonitrile,0.15 w/w of activated carbon was added, and the mixture was stirred at15 to 50° C. for 0.5 to 2 hours. Insoluble substances were removed,followed by addition of 3 volumes of acetonitrile and 1 v/w of pyridine,and then 3.3 v/w of tap water in which 0.426 w/w of sodium periodate wasdissolved. The reaction was allowed to proceed at 15 to 50° C. for 2hours or more. Completion of the reaction was checked by HPLC.

5 v/w of a 20% aqueous sodium thiosulfate solution was added. Insolublesubstances were removed by filtration through celite and washed with 2v/w of ethyl acetate. The filtrate combined with washings wasconcentrated under reduced pressure at 50° C. or less. 10 v/w of ethylacetate and 2 volumes of saturated saline was added to the concentratedsolution, and the organic layer was separated. The organic layer waswashed twice with 2 v/w of saturated saline and further washed with amixture of 4 v/w of 4% aqueous sodium bicarbonate and 4 v/w of saturatedsaline. The organic layer was then dried over an appropriate amount ofmagnesium sulfate. Insoluble substances were removed by filtration andwashed with 3 v/w of ethyl acetate. The filtrate, combined with thewashings, was concentrated under reduced pressure at 50° C. or less.

At a concentration of 10 v/w, 0.122 w/w of sodium acetate and 0.122 w/wof dimethylamine hydrochloride was added and stirred at 0 to 15° C. for15 minutes to 2 hours. 0.316 w/w of sodium triacetoxyborohydride wasadded and stirred at 0 to 40° C. for 1 to 3 hours. Completion of thereaction was checked by HPLC.

The reaction was quenched by adding 7 v/w of a 15% aqueous potassiumhydrogen carbonate solution and 3 v/w of saturated saline. The organiclayer was separated and washed with 5 v/w of tap water and with 3 v/w ofsaturated saline, and dried with an appropriate amount of magnesiumsulfate. Insoluble substances were removed by filtration and the residuewas washed with 3 v/w of ethyl acetate. The filtrate was thenconcentrated under reduced pressure at 50° C. or less to obtain compound(Ia) as the residue. The residue was further purified by chromatographyand/or crystallization.

MS MW 600. HPLC Retention time (RT) 6.2. Purity 90%

Example 19 Alternate Procedure for the Conversion of Compound (III) toCompound (Ia)

2.38 g of acetal at 0° C. and 17 volumes of pyridine/H₂O were mixed,then 5.4 volumes of aqueous KMNO₄ (50 g/L) followed by 3.5 volumes ofaqueous KMNO₄ (50 g/L) were added by slow addition over 30 minutes. Twopeaks on HPLC (RT 9.1 and 9.3 minutes) corresponded to the two possibleisomers at the newly formed hydroxyl group for this reaction. MS MW 603.Conventional extractive work-up gave crude product, 2.86 g. The crudeproduct was used in the following step without further purification.

2.86 g of the above material at room temperature was mixed with 10volumes acetonitrile, 1 volume of pyridine and 1.01 g of NaIO4 in 7.9 mlof water. A complete conversion was observed for the desired compound.MS MW 571. HPLC RT. 9.7. Conventional extractive work-up gave crudeproduct, 2.2 g of material was isolated after the reaction.

For reductive amination, 1.88 g of the above crude aldehyde wasdissolved at 5° C. in 24 ml of EtOAc. 290 mg of NaOAc, followed by 290mg of NMe₂.HCl, followed by 752 mg of Na(AcO)3BH were added to thereaction mixture. Customary extractive work-up at the completion of thereaction gave the crude amino compound (Ia).

Product MS MW 600. HPLC RT. 6.2. Chromatographic purification gave 1 gpure compound (Ia).

Example 20 Conversion of Compound (IV) to Compound (Ib) and Then toCompound (Ia)

0.17 to 7.9 kg of the acetal compound (IV) and 15 v/w of pyridine wereplaced in a reactor and dissolved to a solution, and 2.5 v/w of tapwater was added. The internal temperature was maintained between 25° C.and 55° C. 4.09 v/w of a potassium permanganate solution (50 g per literof water) was added, and the reaction was allowed to proceed for 0.3 to3 hours. The residual amount of acetal compound (IV) was checked byHPLC. 15 v/w of ethyl acetate, 5 v/w of a 10% aqueous citric acidsolution and 3 v/w of saturated saline were added, and the organic layerwas separated. The organic layer was washed with a mixture of 3 v/w of a10% aqueous citric acid solution and 3 v/w of saturated saline, followedby a wash with a mixture of 5 v/w of 4% sodium bicarbonate solution and3 v/w of saturated saline. The product was concentrated under reducedpressure at 50° C. or less to obtain the diol intermediate as a residue.

The diol intermediate residue was dissolved in 7 v/w of acetonitrile,0.15 w/w of activated carbon was added, and the mixture was stirred at15 to 50° C. for 0.5 to 2 hours. Insoluble substances were removed, then3 volumes of acetonitrile and 1 v/w of pyridine were added, followed byaddition of 3.3 v/w of tap water in which 0.426 w/w of sodium periodate(compared to the amount of acetal) was dissolved. The reaction wasallowed to proceed at 15 to 50° C. for 2 hours or more. Completion ofthe reaction was checked by HPLC.

5 v/w of a 20% aqueous sodium thiosulfate solution was added. Insolublesubstances were removed by filtration through celite and washed with 2v/w of ethyl acetate. The filtrate combined with washings wasconcentrated under reduced pressure at 50° C. or less. To theconcentrated solution was added 10 v/w of ethyl acetate and 2 v/w ofsaturated saline. The organic layer was separated and washed twice with2 v/w of saturated saline, followed by a wash with a mixture of 4 v/w of4% aqueous sodium bicarbonate and 4 v/w of saturated saline. The organiclayer was dried ever an appropriate amount of magnesium sulfate, theninsoluble substances were removed and washed with 3 v/w of ethylacetate. The filtrate combined with washings was concentrated underreduced pressure at 50° C. or less. This produced the crude intermediatealdehyde.

To the above concentrate in ethyl acetate (10 v/w compared to theacetal) was added 0.122 w/w of sodium acetate and 0.122 w/w ofdimethylamine hydrochloride. The mixture was stirred at 0 to 15% for 15minutes to 2 hours. 0.316 w/w of sodium triacetoxyborohydride was added,and stirred at 0 to 40° C. for 1 to 3 hours. Completion of the reactionwas checked by HPLC. The reaction was quenched by adding 7 v/w of a 15%aqueous potassium hydrogen carbonate solution and 3 v/w of saturatedsaline. The organic layer was separated, washed with 5 v/w of tap waterand with 3 v/w of saturated saline, and dried with an appropriate amountof magnesium sulfate. Insoluble substances were removed, and the organiclayer was washed with 3 v/w of ethyl acetate and then concentrated underreduced pressure at 50° C. or less to obtain compound (Ib) as theresidue.

The residue (Ib) was dissolved in 8 v/w of ethanol (compared to acetalcompound IV), 0.15 w/w of activated carbon was added, and the mixturewas stirred at 15 to 50° C. for 0.5 to 2 hours. Insoluble substanceswere removed. 5 v/w of ethanol, 2.11 v/w of tap water, 0.5 w/w of 10%Pd/C 50% wet and 0.439 w/w of ammonium formate (compared to acetalcompound IV) were added, and the mixture was stirred under nitrogenatmosphere at 40 to 60° C. for 1 to 4 hours. Completion of the reactionwas checked by HPLC. Upon completion, insoluble substances were removedand washed with 3 v/w of ethanol. The reaction product was thenconcentrated under reduced pressure at 50° C. or less. To theconcentrated residue, 15 v/w of ethyl acetate, 7 v/w of 4% sodiumhydrogen carbonate and 3 v/w of saturate saline were added. The organiclayer was separated and washed with 7 v/w of saturated saline, thendried over an appropriate amount of magnesium sulfate. Insolublesubstances were removed and washed with 3 v/w of ethyl acetate. Thereaction product was then concentrated under reduced pressure at 50° C.or less. Further purification by chromatography and/or crystallizationgave compound (Ia). Product MS. MW 600. HPLC RT. 6.2. Chromatographicpurification gave quality compound (Ia) in 95+% HPLC purity.

Example 21 Conversion of 10-DAB III to Intermediate 10

45 liter of AcOMe was added to the reactor (300 liter), followed by 4.5kg of 10-DAB III and 0.65 kg of malonic acid. To this reaction mixturewas added a solution of N-Bu4NBH4 in AcOMe (4.25 kg of N-Bu4NBH4 inAcOMe 23 L) (reaction temperature: 30-35° C., dropping time: 10-60 min,caution: foaming). A solution of 1.07 kg of malonic acid in 14 liter ofAcOMe was slowly added to the mixture (reaction temperature: 30-35° C.dropping time: 90-150 min). After dropping, the reaction mixture wasmaintained at 30-35° C.

-   -   HPLC conditions:    -   Sample: 0.1 ml of reaction mixture→10 ml/50% aqueous        acetonitrile    -   Injection volume: 1 μl    -   Column: YMC PACK ODS-AM302 (4.6 mm*150 mm, 5 μm)    -   Mobile phase: 0.02M Acetate Buffer (pH 5.0)/acetonitrile (7:3)    -   0.02M Acetate Buffer; 1.36 g of NaOAc→500 ml, the solution was        adjusted to pH 5 by AcOH solution (0.60 g of AcOH→500 ml)    -   Flow rate: 0.7 ml/min    -   Column temperature: 40° C.    -   Detector: UV 230 nm    -   Stop time: 15 min    -   Judgment: 10DAB III not more than 1%

5 L of water was then added to the reaction mixture, with more than 30min of stirring. 23 L of 0.2 N HCl and 23 L of saturated NaCl solutionwere nixed and used for washing the reaction mixture. The extraction wasdone within 10 min. 23 L of 4% NaHCO₃ and 23 L of saturated NaClsolution were mixed and used for washing the reaction mixture. Next, 11L of 4% NaHCO₃ and 11 L of saturated NaCl solution were mixed and usedfor washing the reaction mixture.

22.5 kg of ion exchange resin (Amberlite IRA743) was added to theorganic layer. After more than 1 hour stirring, the mixture was filteredwith a 60 cm Nutsche (SUS) filter. The residue was washed with 45 L ofAcOEt. Combined organic layer was washed with 11 L of saturated NaCl anddried with 2 kg of MgSO₄. After drying, the organic layer was filteredand evaporated under controlled temperature between 20° C. and 40° C.The residue was dissolved with 2.7 liter of methanol and 9.0 liter ofAcOEt, and 36 L of acetonitrile was added under controlled temperaturebetween 20° C. and 30° C. The mixture was stirred slowly, and aprecipitate formed. After not more than 1 hour stirring, the mixture waschilled to between 0° C. and 10° C. After 3 hours, the precipitate wasfiltered with a 60 cm Nutsche (SUS) filter. The precipitate was washedwith 9 L of acetonitrile and dried at a controlled temperature between20° C. and 40° C., producing crystalline intermediate 10. (Standard 3.68kg (Yield 81.5%), Theoretical 4.52 kg. Specification; not less than 70%by HPLC).

-   -   HPLC condition:    -   Sample: 10 mg of sample→10 ml/50% aq. acetonitrile    -   Injection volume: 1 μl    -   Column: YMC PACK ODS-AM302 (4.6 mm*150 mm, 5 μm)    -   Mobile phase: 0.02 M Acetate Buffer (pH 5.0)/acetonitrile (7:3)        0.02M Acetate Buffer; 1.36 g of NaOAc→500 ml, the solution was        adjusted to pH 5 by AcOH solution (0.60 g of AcOH→500 ml)    -   Flow rate: 0.7 ml/min    -   Column temperature: 40° C.    -   Detector: UV 230 nm    -   Stop time: 15 min

Example 22 Conversion of Compound 10 to Intermediate (X) and Then toIntermediate (VII)

10 L of pyridine, 2.91 kg of compound 10 and 2.10 kg of4-dimethylaminopyridine (DMAP) was added to the reactor. The reactionmixture was chilled to between 10° C. and 0° C. under nitrogenatmosphere, and 1.94 kg of trifluoromethansulfonic acid/anhydride wasdropped for between 1 hour and 3 hour. During dropping the reactionmixture was maintained at a temperature between 0° C. and 10° C. Thereaction mixture was monitored by HPLC. After completion of thereaction, 14 L of cyclopentylmethyleter (CPME) was added to the mixtureduring stirring. The precipitate (TFA-DMAP salt) was removed byfiltration using a 30 cm Nutsche filter, and washed with 16 L of CPME.The combined organic layer was first washed with 20 L of water and with20 L of 5% aqueous NaCl solution (twice), followed by washing with 20 Lof 4% NaHCO₃. 10 L of 4% NaHCO₃ and 10 L of saturated NaCl solution weremixed, and the organic layer was washed with this solution. Afterwashing, 2 kg of MgSO₄, 6.0 kg of SiO₂ (florisil), and 4.0 kg of aluminawere added to the organic layer. After drying and de-coloring, theorganic layer was recovered by filtration. The precipitate was washedwith 20 L of CPME. Combined organic layer was evaporated undercontrolled temperatures between 20° C. and 40° C. The obtained residuewas dissolved in 8.0 L of chloroform and heated to a maximum temperatureof 55° C. If the residue did not dissolve completely, several additionalamounts of chloroform were added (up to 5 v/v volume). After slowlychilling to between 30° C. to 20° C., 16 L of IPE and 16 L of n-hexanewere added. After 1 hour stirring at a temperature between 20° C. and30° C. the precipitate was recovered by filtration with a 60 cm Nutsche(SUS) filter. The precipitate was washed with 6 L of IPE/hexane (1:1)solution. After drying with a vacuum drier at temperatures controlledbetween 20° C. to 40° C. for from 3 hours to 72 hours, crystallinecompound VII was obtained. (Standard 1.56 kg (Yield 80.0%), Theoretical1.95 kg, Specification; not leas than 95% by HPLC).

If the product specification was not met, 4 v/v of chloroform was addedto the crude product, and the solution was heated to 55° C. Afterheating, 8 v/v of IPE was added to the solution. After checking forprecipitation, an additional 8 v/v of IPE was slowly added. Afterstirring for from 2 hours to 24 hours, the precipitated product compoundVII was obtained by filtration. (yield 80-85%). This step can berepeated until product purity meets specifications.

Example 23 Conversion of Compound Intermediate VII to Intermediate IV

15 L of ethyl acetate, 1.8 kg of alcohol compound VII, 0.23 kg oftriethylamine HCl salt (TEA.HCl), and 1.3 kg of acrolein diethyl acetal(ADA) was added to the reactor. 7 g of camphorsulfonic acid (CSA) wasdissolved in 1 L of AcOEt, and the solution was added to the reactionmixture, maintaining the temperature between 15° C. and 25° C. forseveral hours (from 4 hours to 28 hours). The reaction mixture waschecked by HPLC for completion. Upon completion, 15 L of isopropyl ether(IPE) and 15 L of n-hexane was added. The mixture was chilled to between10° C. and 0° C., and stirred for from 1 to 3 hours while maintainingthe temperature. After stirring, the precipitate was obtained byfiltration using a 60 cm Nutsche filter. The precipitate (crude acetalwet) was washed with 15 L of IPE and dissolved in 30 L of AcOEt. Theorganic layer was washed with 15 L of water, and twice with 9 L ofwater. Next, 5 L of 4% NaHCO₃ and 5 L of saturated NaCl solution weremixed and used to wash the organic layer. The organic layer was thendried with 1 kg of MgSO₄. It was filtered and the residue was washed 9liter of AcOEt.

The combined organic layer was evaporated under controlled temperaturebetween 20° C. and 40° C. until the residue was between 1.5 w/w and 2.0w/w. 11 L of IPE was added to the residue, and the organic layer wasstirred under controlled temperature between 20° C. and 30° C. 11 L ofn-hexane was added to the mixture, and the mixture was stirred for notless than 1 hour. After chilling to between 10° C. and 0° C., themixture was stirred for not less than 1 hour. The precipitate wasfiltered with a 60 cm Nutsche (SUS) filter. The precipitate was washedwith 5 L of IPE. After drying with vacuum drier at 20° C. and 40° C.,crystalline acetal compound IV was obtained. 1.0 kg, Yield 50%.

All publications cited in the specification, both patent and non-patentpublications, are indicative of the level of skill of those skilled inthe art to which this invention pertains. All these publications areherein fully incorporated by reference to the same extent as if eachindividual publication were specifically and individually indicated asbeing incorporated by reference.

1-17. (canceled)
 18. A method of producing an acid salt of tesetaxel,comprising: adding an acid selected from a monobasic acid, a dibasicacid, a tribasic acid, or a polybasic acid to a solution of tesetaxel inan organic solvent, thereby forming a mixture; and crystallizing theacid salt of tesetaxel from the mixture; provided that either: (a) thesolvent is isopropyl alcohol, or (b) the acid is selected from benzoicacid, ortho-toluic acid, meta-toluic acid, para-toluic acid, ethanesulphonic acid, camphor sulphonic acid, (S)-(+)-mandelic acid,(R)-(−)-mandelic acid, gentisic acid, hippuric acid, glycolic acid,2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, gluconic acid,malic acid, oxalic acid, succinic acid, tartaric acid, malonic acid,adipic acid, itaconic acid, cyclohexane dicarboxylic acid, phthalicacid, edisylate, phenyl phosphonic acid, digluconic acid, dibasicnatural amino acids, or phosphoric acid.
 19. The method of claim 18,wherein the acid is selected from benzoic acid, ortho-toluic acid,meta-toluic acid, para-toluic acid, ethane sulphonic acid, camphorsulphonic acid, (S)-(+)-mandelic acid, (R)-(−)-mandelic acid, gentisicacid, hippuric acid, glycolic acid, 2-hydroxyethanesulfonic acid,2-naphthalenesulfonic acid, gluconic acid, malic acid, oxalic acid,succinic acid, tartaric acid, malonic acid, adipic acid, itaconic acid,cyclohexane dicarboxylic acid, phthalic acid, edisylate, phenylphosphonic acid, digluconic acid, dibasic natural amino acids, orphosphoric acid.
 20. The method of claim 18, wherein the acid isselected from benzoic acid, ortho-toluic acid, meta-toluic acid,para-toluic acid, ethane sulphonic acid, camphor sulphonic acid,(S)-(+)-mandelic acid, (R)-(−)-mandelic acid, gentisic acid, hippuricacid, glycolic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonicacid, and gluconic acid.
 21. The method of claim 18, wherein the acid isselected from malic acid, oxalic acid, succinic acid, tartaric acid,malonic acid, adipic acid, itaconic acid, cyclohexane dicarboxylic acid,phthalic acid, edisylate, phenyl phosphonic acid, digluconic acid, anddibasic natural amino acids.
 22. The method of claim 18, wherein theacid is phosphoric acid.
 23. The method of claim 18, wherein the organicsolvent is selected from acetone, methanol, ethanol, propanol, butanol,acetonitrile, tetrahydrofuran, isopropyl alcohol, toluene andN,N-dimethylformamide.
 24. The method of claim 23, wherein the organicsolvent is isopropyl alcohol.
 25. A method of producing an acid salt oftesetaxel, comprising: adding an acid selected from a monobasic acid, adibasic acid, a tribasic acid, or a polybasic acid to a first mixturecomprising an organic solvent and a compound represented by formula(Ia):

linking the compound of Formula (Ia) with a taxane side chain precursor,thereby producing a second mixture comprising 2′-O protected tesetaxel;deprotecting the 2′-O protected tesetaxel group, thereby producing athird mixture comprising tesetaxel; and crystallizing the acid salt oftesetaxel from the third mixture.
 26. The method of claim 25, whereinthe acid is selected from benzoic acid, ortho-toluic acid, meta-toluicacid, para-toluic acid, ethane sulphonic acid, camphor sulphonic acid,(S)-(+)-mandelic acid, (R)-(−)-mandelic acid, gentisic acid, hippuricacid, glycolic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonicacid, gluconic acid, malic acid, oxalic acid, succinic acid, tartaricacid, malonic acid, adipic acid, itaconic acid, cyclohexane dicarboxylicacid, phthalic acid, edisylate, phenyl phosphonic acid, digluconic acid,dibasic natural amino acids, or phosphoric acid.
 27. The method of claim25, wherein the acid is selected from benzoic acid, ortho-toluic acid,meta-toluic acid, para-toluic acid, ethane sulphonic acid, camphorsulphonic acid, (S)-(+)-mandelic acid, (R)-(−)-mandelic acid, gentisicacid, hippuric acid, glycolic acid, 2-hydroxyethanesulfonic acid,2-naphthalenesulfonic acid, and gluconic acid.
 28. The method of claim25, wherein the acid is selected from malic acid, oxalic acid, succinicacid, tartaric acid, malonic acid, adipic acid, itaconic acid,cyclohexane dicarboxylic acid, phthalic acid, edisylate, phenylphosphonic acid, digluconic acid, and dibasic natural amino acids. 29.The method of claim 25, wherein the acid is phosphoric acid.
 30. Themethod of claim 25, wherein the organic solvent is selected fromacetone, methanol, ethanol, propanol, butanol, acetonitrile,tetrahydrofuran, isopropyl alcohol, toluene and N,N-dimethylformamide.31. The method of claim 30, wherein the organic solvent is isopropylalcohol.